A ratiometric fluorescent probe for detecting cadaverine and a preparation method and application thereof

Abstract

The application discloses a ratio type fluorescent probe for detecting cadaverine as well as a preparation method and application of the probe, the probe can respond to cadaverine in 50% DMSO solvent, the fluorescence of the probe at 465 nm is quenched, and the fluorescence at 402 nm is enhanced with the increase of the cadaverine concentration. The probe PCN has the characteristics of quick response (about 35s), meanwhile, the probe has good selectivity and anti-interference, can effectively exclude the interference of other biological amines in food matrix, the probe molecule disclosed by the application can not only qualitatively and quantitatively detect cadaverine in real samples, but also has good application in paper-based and gel detection, and has great application potential in visualized fluorescence detection of gaseous and liquid cadaverine and visualized quantitative fluorescence detection of the freshness of actual food samples.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection, and particularly relates to a ratiometric fluorescent probe for detecting cadaverine, its preparation method, and its application. Background Technology

[0002] Biogenic amines are basic nitrogenous compounds produced during food spoilage. They are generated by the decarboxylation of amino acids during microbial activity or endogenous tissue metabolism and are commonly found in high-protein foods such as meat. There are eight common biogenic amines in food: spermine, putrescine, cadaverine, tryptamine, phenylethylamine, spermidine, histamine, and tyramine. Cadaverine is generally considered a key compound indicating the degree of microbial decay in meat and can be used to assess food freshness. Ingesting high concentrations of cadaverine can lead to headaches, bronchitis, and pseudo-allergic reactions, and even food poisoning and other health hazards. Cadaverine in food can react with nitrites to produce carcinogenic nitrosamines, and putrescine and cadaverine can also increase the toxicity of histamine in food. Given the physiological and toxicological effects of cadaverine, and the fact that spoiled food usually contains high concentrations of cadaverine, detecting food safety issues caused by cadaverine is crucial.

[0003] Currently, there are various analytical techniques for detecting cadaverine, such as gas chromatography-mass spectrometry, high-performance liquid chromatography, electrochemical detection, and Raman spectroscopy. However, most of these methods are complex to operate, require expensive instruments, and cannot meet the requirements for real-time on-site detection. Fluorescence spectroscopy, on the other hand, has advantages such as high sensitivity, good specificity, and fast response time, making it one of the most promising methods for rapid on-site detection of cadaverine. Researchers have reported some fluorescent probes for detecting cadaverine, but most of these probes are based on a single change in fluorescence intensity signal, making them susceptible to interference from background fluorescence. A few ratiometric fluorescent probes suffer from slow response speeds and cumbersome synthesis processes, limiting their ability for rapid, visualized, and real-time detection. Therefore, it is essential to develop a simple, highly selective, and fast-responding fluorescent probe for the detection of cadaverine. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a ratiometric fluorescent probe for detecting cadaverine, its preparation method, and its application. The organic fluorescent small molecule probe provided by this invention can achieve rapid and visual detection of cadaverine and effectively eliminate interference from various other biogenic amines. Furthermore, by combining smartphones with the extraction of RGB values ​​from fluorescence images based on paper-based and gel fluorescence sensors, it is possible to further achieve visual and quantitative detection of the freshness of actual beef and shrimp samples, providing a simple, rapid, and accurate method for assessing food freshness.

[0005] To achieve the above-mentioned objective, the present invention provides a fluorescent probe for detecting cadaverine, with the following structure:

[0006]

[0007] The method for preparing the ratiometric fluorescent probe for detecting cadaverine includes the following steps:

[0008] 1-Pyrene formaldehyde, malondicyandiamide, and piperidine were placed in a reaction flask, anhydrous ethanol was added, and the mixture was stirred at room temperature for 5 hours. After the reaction, the solvent was evaporated to dryness to obtain a solid crude product, and the probe PCN was obtained by column chromatography.

[0009] Furthermore, the molar ratio of 1-pyrene formaldehyde to malondicyandiamide is 1:3; the column chromatography uses 200-300 mesh silica gel, and the eluent is V... 二氯甲烷 / V 石油醚 =1 / 1.

[0010] Its synthetic route is:

[0011]

[0012] Specific synthesis method: 1-Pyrenecarboxaldehyde (0.92 g, 4 mmol), malondicyandiamide (0.79 g, 12 mmol), and piperidine (1 mL) were placed in a flask, and 20 mL of anhydrous ethanol was added. The mixture was stirred at room temperature for 5 hours. After the solvent was evaporated under vacuum from the reaction mixture, the orange solid probe PCN was purified by column chromatography using dichloromethane:petroleum ether = 1:1 as the eluent.

[0013] The mechanism of the probe of this invention is as follows:

[0014]

[0015] The mechanism of action of the fluorescent probe of this invention is as follows: the cyano group, as an electron-withdrawing group, can reduce the electron density of the carbon-carbon double bond, thereby allowing cadaverine to easily attack the carbon-carbon double bond, interrupting the π-π conjugated structure of the probe, and changing the fluorescence signal by altering the electronic configuration, accompanied by a blue shift in fluorescence. The detection system exhibits high selectivity for cadaverine, rapid response (~35s), and low detection limit (31nM).

[0016] The application of the ratiometric fluorescent probe for detecting cadaverine in the qualitative and quantitative detection of cadaverine.

[0017] The application specifically includes: dissolving the fluorescent probe PCN in DMSO solvent to prepare a 1×10⁻⁶ solution. -3 A probe solution of mol / L was prepared, and then the analyte was added. After the reaction, the fluorescence changes at 402 nm and 465 nm were observed and recorded by fluorescence detection.

[0018] The application specifically includes: dissolving the fluorescent probe PCN in DMSO to prepare a concentration of 1.0 × 10⁻⁶ PCN. -5 A solution of mol / L was prepared, and then the sample to be tested was added. The fluorescence intensity was detected, and the content of cadaverine was quantitatively calculated based on the linear relationship between the fluorescence intensity and the concentration of the sample to be tested.

[0019] The application described uses paper-based detection of cadaverine. The specific steps are as follows: cut experimental filter paper into strips of the same size and soak them in a solution of 1.0 × 10⁻⁶ mol / L. -4 In a mol / L PCN solution, for liquid cadaverine, different equivalents of cadaverine solution were added after the test strip dried, and the changes in fluorescence intensity were observed. For gaseous cadaverine, the test strip was loaded onto the cap of a vacuum sample bottle, and different concentrations of cadaverine were mixed with dichloromethane in the bottle. The mixture was heated to allow it to fully evaporate, and the changes in fluorescence intensity were observed.

[0020] The application described uses gel electrophoresis to detect cadaverine. The specific steps are as follows: For liquid cadaverine, prepare a solution with a concentration of 1.0 × 10⁻⁶. -4 PCN gels of mol / L were immersed in cadaverine solutions of different equivalents, and the changes in fluorescence intensity were observed. For gaseous cadaverine, the gel was loaded onto the cap of a vacuum sample vial, and different concentrations of cadaverine were mixed with dichloromethane in the vial. The mixture was heated to allow it to fully evaporate, and the changes in fluorescence intensity were observed.

[0021] The beneficial effects of this invention are: the ratiometric fluorescent probe of this invention has a simple synthetic route, rapid response to cadaverine, and can distinguish other structurally similar biogenic amines, exhibiting excellent selectivity and anti-interference ability. Furthermore, by combining the fluorescence images of the probe-loaded paper-based and gel fluorescent sensors with a smartphone for RGB analysis, the RGB values ​​of the fluorescence images can be output in real time. This not only enables quantitative detection of gaseous and liquid cadaverine but also achieves visualized quantitative detection of the freshness of actual beef and shrimp samples. Attached Figure Description

[0022] Figure 1 The image shows the proton NMR spectrum of the probe PCN in deuterated DMSO, with the x-axis representing chemical shift (ppm) and the y-axis representing intensity.

[0023] Figure 2 The image shows the carbon spectrum of the probe PCN in deuterated DMSO, with the horizontal axis representing chemical shift (ppm) and the vertical axis representing intensity.

[0024] Figure 3 The mass spectrometry of the probe PCN in this invention uses ESI as the ionization source. + ;

[0025] Figure 4 The probe PCN (1×10) in this invention-5 The fluorescence spectrum of cadaverine solution reacting with cadaverine solution in 50% DMSO solution at room temperature over time; excitation wavelength: 360 nm;

[0026] Figure 5 The probe PCN (1×10) in this invention -5 mol / L, in 50% DMSO solution, reacted at room temperature with biogenic amines (cadaverine, putrescine, spermine, spermidine, tyramine, histamine), organic amines (ethylamine, n-propylamine, hydroxylamine), inorganic amines (hydrazine hydrate), and inorganic salt ions (Mg²⁺). 2+ Zn 2+ NO3 - SO4 2- Fluorescence spectra of bioactive substances (cysteine, glutathione, glucose) and cadaverine were recorded; excitation wavelength: 360 nm;

[0027] Figure 6 This is a graph showing the fluorescence intensity and linear relationship between the probe PCN and different concentrations of cadaverine in this invention.

[0028] Figure 7 The figures show the visualized detection results of liquid and gaseous cadaverine using the probe paper base of this invention, as well as the visualized quantitative detection results of liquid and gaseous cadaverine using RGB analysis.

[0029] Figure 8 The figures show the visualized detection results of the probe gel of this invention on liquid and gaseous cadaverine, as well as the visualized quantitative detection results of liquid and gaseous cadaverine combined with RGB analysis.

[0030] Figure 9 This is a visualization of the detection results of the probe paper base of the present invention on the freshness of beef at different temperatures;

[0031] Figure 10 This is a visualization of the detection results of the probe paper base of the present invention on the freshness of shrimp at different temperatures;

[0032] Figure 11 This is a comparison chart of the visual detection results of the probe gel of the present invention on the freshness of beef, the visual quantitative detection results of gaseous cadaverine combined with RGB analysis, and the TVBN value of the national standard method.

[0033] Figure 12 This invention presents a visual detection result of shrimp freshness using probe gel, a visual quantitative detection result of gaseous cadaverine using RGB analysis, and a comparison chart of TVBN values ​​using the national standard method.

[0034] Figure 13 The mass spectrum of PCN-Cad in this invention is shown. The ionization source is ESI. + . Detailed Implementation

[0035] The present invention will be further described below with reference to examples and accompanying drawings, but the present invention is not limited to the following embodiments.

[0036] Example 1: Synthesis of PCN fluorescent probe

[0037] In a 50 mL round-bottom flask, 1-pyrenecarboxaldehyde (0.92 g, 4 mmol), malondicyandiamide (0.79 g, 12 mmol), and piperidine (1 mL) were added, along with 20 mL of anhydrous ethanol. The mixture was stirred at room temperature for 5 hours. After the solvent was evaporated under vacuum, the reaction mixture was purified by column chromatography using dichloromethane:petroleum ether = 1:1 as the eluent to obtain an orange solid probe PCN (0.70 g, yield 62.95%). Figure 1 This is the proton NMR spectrum of the probe. 1 H NMR (DMSO-d6, 400MHz) δ9.63 (s, 1H), 8.71 (dd, J = 12.72, 8.24Hz, 2H), 8.53–8.42 (m, 5H), 8.31 (d, J = 8.92Hz, 1H), 8.22 (t, J = 7.64Hz, 1H). Figure 2 The carbon NMR spectrum of the probe. 13 C NMR(DMSO-d6,100MHz)δ159.56,135.25,131.26,131.24,131.08,130.54,130.51,130.12,127 .95,127.76,127.69,126.40,125.55,125.17,124.13,123.61,123.34,115.04,114.40,83.66. Figure 3 For the probe's mass spectrometry, ESI-Mass: calcd for C 20 H 10 N2:278.0844,found[M+H] + at m / z 279.0926.

[0038] Example 2: Response time of PCN fluorescent probe to cadaverine

[0039] The fluorescent probe PCN was dissolved in DMSO solvent to prepare a solution of 1×10⁻⁶. -3 A probe solution of mol / L was prepared. 30 μL of the probe solution was added to 1470 μL of DMSO solution and 900 μL of ultrapure water, followed by the addition of 600 μL of cadaverine. After the reaction, the change in the ratio of the probe's fluorescence intensity at 402 nm and 465 nm was recorded using fluorescence detection. Figure 4 It can be seen that the fluorescence intensity ratio of the probe after the addition of cadaverine (I)402 / I 465 The fluorescence intensity increases rapidly, reaching a stable level after 35 seconds.

[0040] Example 3: Selectivity of PCN fluorescent probes for different biogenic amines

[0041] The PCN fluorescent probe in Example 1 was configured as 1×10 -3 mol / L of mother liquor.

[0042] Prepare a solution of the following substances: cadaverine, putrescine, spermine, spermidine, tyramine, histamine, ethylamine, n-propylamine, hydroxylamine, hydrazine hydrate, cysteine, glutathione, glucose, MgCl2·6H2O, ZnCl2, NaNO3, and Na2SO4, to a concentration of 1×10⁻⁶. -3 mol / L of mother liquor.

[0043] Take 17 test tubes and add 300 μL of the different biogenic amines and other interfering stock solutions to each tube, followed by 1200 μL of ultrapure water, then 1470 μL of DMSO solution, and finally 30 μL of probe stock solution to each tube. After mixing all solutions, perform fluorescence detection (Ex = 360 nm). Plot fluorescence intensity on the ordinate and wavelength on the abscissa. Figure 5 .Depend on Figure 5 It can be observed that cadaverine can enhance the fluorescence of the PCN probe, while other biogenic amines and interfering ions have almost no effect on its fluorescence intensity.

[0044] Example 4: Fluorescence intensity of PCN at different concentrations of cadaverine

[0045] The concentration is 1×10 -3 A mol / L cadaverine stock solution was used, and the final cadaverine concentration was controlled by varying the volume added to different test tubes, resulting in cadaverine contents ranging from 0 to 20 equivalents. After the reaction, fluorescence detection (Ex = 360 nm) was performed to measure the fluorescence intensity in each system. The fluorescence intensity ratio (F...) was then expressed as... 402 / F 465 Plot a curve with γ as the ordinate and cadaverine concentration as the abscissa, and draw a linear relationship. Figure 6 It can be seen that as the concentration of cadaverine increases, the fluorescence of the probe is quenched at 465 nm, and the fluorescence intensity at 402 nm gradually increases. When the content of cadaverine reaches 20 equivalents, the fluorescence of the probe no longer increases.

[0046] Example 5: Detection of cadaverine in different food samples using PCN fluorescent probes

[0047] Three food samples (beef, fish, and pork) were cut into small pieces and homogenized. 1g of each food sample and 60mL of ethanol were placed in an Erlenmeyer flask and sonicated for 5 minutes. The mixture was then centrifuged at 10,000 rpm for 10 minutes. After cooling to room temperature, the supernatant was collected, and a certain amount of PBS and NaOH solution was added to maintain the pH at 7.4. Finally, 30μL of PCN stock solution and different concentrations of cadaverine solutions (20, 40, 60, 80, 100μM) were added to test tubes, followed by the food sample solution and DMSO, adjusting the total volume to 3mL (DMSO / food sample solution = 1 / 1, v / v). After mixing all solutions, fluorescence detection (Ex = 360nm) was performed, and the recovery rate and RSD were calculated. Table 1 is plotted with the actual sample as the ordinate and cadaverine concentration, recovery rate, and RSD as the abscissa.

[0048] Table 1: Data on the recovery rate of cadaverine in actual food samples

[0049]

[0050] Example 6: Application of PCN probe in detecting liquid and gaseous cadaverine on paper substrates

[0051] Completely immerse square filter paper strips of the same size in the probe stock solution (c = 1 × 10⁻⁶). -4 The solution was prepared in mol / L solution and then dried in air. When detecting liquid cadaverine using the PCN probe paper-based device, cadaverine solutions of different concentrations were directly dropped onto the paper base, and the fluorescence color change was recorded using a digital camera under a handheld UV lamp (365nm). When detecting gaseous cadaverine using the PCN probe paper-based device, 10mL sample vials containing cadaverine solutions of different concentrations were tightly capped and gently heated for a period of time to obtain a sealed cadaverine gas atmosphere. The PCN probe paper base was then suspended above the sample vials, fully exposing them to cadaverine vapor. After the color change was complete, the fluorescence color change was recorded using a digital camera under a handheld UV lamp (365nm).

[0052] Example 7: Application of PCN probe in detecting liquid and gaseous cadaverine on gels

[0053] Dissolve 2.0 g of sodium alginate in 50 mL of PBS buffer (pH = 7.4, 1 × 10⁻⁶). -2 In a solution of mol / L, the mixture was stirred vigorously at room temperature for 3 hours. Then, 5 mL of PCN probe stock solution (1×10⁻⁶ mol / L) was added. -2 The PCN gel precursor solution (1×10⁻⁶ mol / L) was obtained by dissolving 45 mL of DMSO in 45 mL of DMSO. -4(mol / L, PBS / DMSO = 1 / 1, v / v). The solution was then sonicated for 20 min to remove air bubbles, and allowed to stand overnight at 4°C to remove further bubbles. The gel precursor solution was dripped into a slowly stirred CaCl2 solution (5%, w / v, 100 mL) using a syringe to form uniformly shaped small beads. After soaking for 5 minutes, the gel beads were separated and washed with deionized water for subsequent testing. The testing procedure was the same as in Example 5.

[0054] Example 8: Colorimetric Data Analysis

[0055] Fluorescence photographs of paper substrates and gels exposed to different concentrations of cadaverine in Example 6 were obtained by using color analysis software to output RGB values ​​in real time. A standard curve was obtained by linear fitting with liquid / gaseous cadaverine concentration as the x-axis and G / G0 as the y-axis. Figure 7 and Figure 8 The results showed that G / G0 had a good linear relationship with both liquid and gaseous cadaverine concentrations. This indicates that by combining a smartphone with the data, fluorescence images can be converted into RGB values ​​in real time, which can then be substituted into a standard curve to obtain the corresponding cadaverine concentration, thus achieving the goal of visual quantitative detection of cadaverine. This method does not require expensive instruments, can effectively eliminate the color discrimination error of the human eye through digital means, and has the advantages of being portable, fast, and accurate.

[0056] Example 9: Application of portable PCN probe device for real-time food monitoring

[0057] Cut beef and shrimp samples were placed separately in petri dishes along with paper base and gel, sealed with plastic wrap, and stored at room temperature (25°C), refrigerated (0°C), and frozen (-25°C). The paper base and gel were removed from the petri dishes at regular intervals for observation, and changes were recorded using a digital camera under a UV lamp (365nm). Figure 9 and Figure 10 As shown, the paper substrate exhibits significant fluorescence color changes in monitoring the freshness of beef and shrimp samples stored at different temperatures. It can be seen that for the two foods stored at 25℃, the paper substrate color changes from dark red to purple and finally to bright green over time. The color change of the paper substrate stored at 0℃ is similar to that at 25℃. At -25℃, the paper substrate color gradually darkens from red, indicating that the two foods produce less cadaverine under these conditions, and low temperature is more conducive to food storage.

[0058] The RGB values ​​of gel fluorescence images at different temperatures and times can be output in real time via a smartphone. A fitting curve is plotted with G / G0 on the ordinate and storage time on the abscissa, and the results are compared with the total volatile basic nitrogen (TVBN) value determined by the national standard method. Figure 11 , Figure 12As shown in the figure, the cadaverine content produced during food spoilage, as measured by the PCN probe gel, is basically consistent with the value determined by the national standard method. This indicates that the PCN probe can achieve visualized quantitative detection of the freshness of actual food samples and saves a significant amount of detection time.

Claims

1. The application of a ratiometric fluorescent probe for detecting cadaverine in the qualitative and quantitative detection of cadaverine, characterized in that, The ratiometric fluorescent probe has the following structural formula: 。 2. The application of the ratiometric fluorescent probe for detecting cadaverine according to claim 1, characterized in that, Specifically, it includes: The fluorescent probe PCN was dissolved in DMSO solvent to prepare a solution of 1 × 10⁻⁶. -3 A probe solution of mol / L was prepared, and then the analyte was added. After the reaction, the fluorescence changes at 402 nm and 465 nm were observed and recorded by fluorescence detection.

3. The application of the ratiometric fluorescent probe for detecting cadaverine according to claim 1, characterized in that, Specifically, it includes: The fluorescent probe PCN was dissolved in DMSO to prepare a solution with a concentration of 1.0 × 10⁻⁶. -5 A solution of mol / L was prepared, and then the sample to be tested was added. The fluorescence intensity was detected, and the content of cadaverine was quantitatively calculated based on the linear relationship between the fluorescence intensity and the concentration of the sample to be tested.

4. The application of the ratiometric fluorescent probe for detecting cadaverine according to claim 1, characterized in that, The paper-based method for detecting cadaverine involves the following steps: cutting experimental filter paper into strips of the same size and soaking them in a solution of 1.0 × 10⁻⁶ mol / L. -4 In a mol / L PCN solution, for liquid cadaverine, different equivalents of cadaverine solution were added after the test strip dried, and the changes in fluorescence intensity were observed. For gaseous cadaverine, the test strip was loaded onto the cap of a vacuum sample bottle, and different concentrations of cadaverine were mixed with dichloromethane in the bottle. The mixture was heated to allow it to fully evaporate, and the changes in fluorescence intensity were observed.

5. The application of the ratiometric fluorescent probe for detecting cadaverine according to claim 1, characterized in that, The gel electrophoresis method was used to detect cadaverine. The specific steps were as follows: For liquid cadaverine, a concentration of 1.0 × 10⁻⁶ was prepared. -4 PCN gels of mol / L were immersed in cadaverine solutions of different equivalents, and the changes in fluorescence intensity were observed. For gaseous cadaverine, the gel was loaded onto the cap of a vacuum sample vial, and different concentrations of cadaverine were mixed with dichloromethane in the vial. The mixture was heated to allow it to fully evaporate, and the changes in fluorescence intensity were observed.

Images